Differentiation (2000) 66:93–105 C Blackwell Wissenschafts-Verlag 2000 ORIGINAL ARTICLE Clotilde Gimond ¡ Christian Baudoin Arnoud Sonnenberg Defects in adhesion and migration, but not in proliferation and differentiation, of embryonic stem cells upon replacement of integrin subunit b1A by b1D Accepted in revised form: 7 June 2000 Abstract b1D is a skeletal muscle-specific splice variant of the b1 integrin subunit, while b1A integrin subunit has a wide tissue distribution. We have previously shown that replacement of b1A by b1D by homologous recom- bination (knockin) in all mouse tissues was embryonic lethal. Through two successive rounds of homologous recombination, we have now produced embryonic stem (ES) cells expressing b1D instead of b1A, and analyzed the ability of b1D to support ES cell differentiation in vitro and in teratomas in vivo. b1D knockin (KI) ES cells grew at a similar rate but as more compact colonies than the b1A-expressing cells. Increased cell cohesive- ness, however, did not appear to involve changes in cadherin activity. Although in both b1A and b1D-KI ES cells only one b1 allele is active, the expression of b1 integrins in the b1D-KI ES cells was reduced by 50%, compared with that in the b1A-expressing cells; this cor- related with impaired adhesive and migratory capacities. It appeared that during in vitro cardiac differentiation, in spite of a slight delay in the induction of two cardiac- specific transcripts, the a- and b-myosin heavy chains, contracting cardiomyocytes were detected in similar numbers and at the same time in embryoid bodies (EB) derived from b1D-KI and from b1A cells. Furthermore, replacement of b1A by b1D in ES cells did not affect C. Gimond 1 ¡ C. Baudoin 1 ¡ A. Sonnenberg ( ✉ ) The Netherland Cancer Institute, Division of Cell Biology, Plesmanlaan 121, NL-1066 CX Amsterdam, The Netherlands e-mail: asonn/nki.nl Tel: π 31 20 512 1942, Fax: π 31 20 512 1944 Present address: 1 Institut de Signalisation, Biologie du De ´veloppment et Cancer, CNRS-UMR 6543 – Centre A. Lacassagne, 33 Ave de Valombrose, 06189 Nice Cedex, France 2 Faculte ´ de Me ´dicine, INSERM U385, 28 Ave de Valombrose, 06107 Nice Cedex 02, France U.S. Copyright Clearance Center Code Statement: 0301–4681/2000/6602–93$ 15.00/0 neurite differentiation in embryoid bodies in the pres- ence of retinoic acid suggesting that b1D supports neurogenesis. However, the impaired migration of other cells from the EB, including endodermal cells, prevented the normal outgrowth of neurites in b1D-KI EB. Fi- nally, injection of b1D-KI ES cells in the flank of syn- geneic mice gave rise to fully developed teratomas con- taining simple and pluristratified epithelia, muscle, carti- lage, blood vessels, and tissues from the neural lineage. These results show that the muscle-specific splice variant b1D, in spite of its specific cytoplasmic domain, sup- ports the differentiation of many cell types. This further suggests that the embryonic lethality in the b1D-KI em- bryos was mainly due to the different ability of b1A and b1D to mediate cell adhesion and migration. Key words b1 integrin ¡ splice variant ¡ embryonic stem cells ¡ migration ¡ proliferation ¡ differentiation Introduction Integrins are a large family of heterodimeric receptors involved in cell-cell adhesion and in binding of cells to the ECM proteins. They are formed by non-covalently associated a and b subunits, each composed of an extra- cellular, a transmembrane, and a cytoplasmic domain and provide a link between the ECM and the cytoskel- eton of the cell (reviewed in [20]). 18 a and 8 b subunits have been identified which constitute a family of over 20 distinct receptors. The b1 subunit associates with vari- ous a chains to form the most abundantly and ubiqui- tously expressed subfamily of integrins. Antibody inhi- bition studies and disruption of the b1 subunit gene in vivo by homologous recombination have demonstrated the critical role of b1 integrins in early development, cell differentiation and migration, hematopoiesis, angiogen- esis, and assembly of extracellular matrix proteins [10].